利用酵母双杂交技术筛选鉴定STCH与RanBP9的相互作用

以人应激和分子伴侣蛋白(STCH)为诱饵蛋白,利用酵母双杂交技术在高严格条件下筛选人脑cDNA文库。在SD/-4培养基上共获得156个阳性克隆,将酵母菌扩增后抽提质粒,转化大肠杆菌,进行序列分析,并经过回复杂交验证,潜在的与STCH相互作用的蛋白有:VDAC、MBP2、ZNF251、RanBP9、Phosophate glycolase、β-tubulin、up1、up2和WW adaptor蛋白。基于各候选蛋白与神经系统疾病的关系,选择RanBP9作进一步验证实验。经体外GST-Pulldown和体内免疫共沉淀实验证实RanBP9和STCH能够相互作用,明确RanBP9的羧基端为相互作用的最小功能域。此结果为我们进一步揭示STCH作用的分子机制提供了线索。

Screening and identification of the interaction of STCH and RanBP9 by yeast two-hybrid system

Under high stringent selection condition, yeast two-hybrid technique was performed to screen a human brain cDNA library with stress and chaperone (STCH) as a bait protein. One hundred and fifty-six positive clones were obtained from SD/-4 medium plate. The plasmids from yeast were transformed into Ecoli DH5α and the plasmid from Ecoli was extracted for sequencing. After repetitive hybrid confirmation, the putative proteins that can interact with STCH include VDAC, MBP2, ZNF251, RanBP9, Phosophate glycolase, β-tubulin, up1, up2 and WW adaptor proteins. Based on their functions in neurological diseases, RanBP9 was selected for further validation. The interaction of STCH and RanBP9 was confirmed by in vitro GST-Pulldown and in vivo co-immunoprecipitation, and the minimum domain of RanBP9 interacting with STCH was mapped to carboxyl terminal. These data provide useful clues for elucidating the molecular mechanism of the function of STCH.